An antibody medicament, which is based on the mechanism of biological protection in human, is highly expected since it is a molecular targeting therapeutics which targets a specific functional molecule. Its high efficacy shown in the field of cancer and rheumatism, inter alia, has boosted the world-wide development rush in recent years and its world market scale is thought to keep magnifying for the time being.
Under such social background, marked progress in the technique has been developed in the field of an antibody medicament and antibody engineering. However, some of the obtained clones of antibody molecules have posed much difficulty in their handling due to a low level of expression and/or stability and its solution is not so easy a matter. The problems stated hereinabove have been a great burden to research and development with an antibody.
Another problem confronted in case of an antibody medicament is its cost. Since a comparatively large quantity of antibodies is required for the therapy and also heavy investment in plant and equipment occurs, increase in not only a burden of patients but also in national expenses for medical treatment will result. Accordingly, an important proposition for the development of an antibody medicament is to improve productivity and to lower its cost.
In the living body, an antibody sequence is produced by a random genetic recombination or mutagenesis occurring while maturation of B cells, among which one having an optimized antibody sequence selected and proliferated. The optimization chiefly depends upon an antigen-binding capacity. However, a number of factors including stability of domains, interaction between H chain and L chain, interaction between a variable region and a constant region, a protease sensibility and secretion efficiency are thought to complicatedly attribute to the optimization. Thus, natural antibody sequences are not necessarily optimized with respect of stability.
If an isolated antibody clone is expressed as a recombinant protein for analysis, it is sometimes found to be fairly unstable. As a consequence, various problems arise, i.e. (1) an expression level is extremely low; (2) it is expressed not in a soluble form but as inclusion bodies to thereby necessitate refolding; (3) exposure to an acid while purification causes denaturation; or (4) precipitation and denaturation occurs with standing at room temperature or even at 4° C. and thus the reactivity disappeared.
For resolving these problems, the first approach may be investigation of optimal conditions of operations or buffers for respective antibody clones. However, the investigation would not only require much labor and cost but in some cases, by failure of dissolving the problems, might drive a research worker into giving-up of the subsequent analysis or development of an antibody clone which may have had a promising reactivity.
The second approach may be a molecular modification such as amino acid substitution or conversion of a molecular form for aiming at the improvement of the properties of an antibody. In general, modification for improving protein stability includes transplantation more conserved amino acids from other homologous sequences, and rational design via computer modeling for increasing hydrophilicity on the molecular surface or increasing hydrophobicity within the interior of hydrophobic core to enhance the strength of packing (Non-patent reference 1).
With respect to the improvement of stability or an expression level of an antibody molecule via amino acid substitution, many of previous reports are concerned with characteristic amino acid modification for sequences of respective antibody clones but there are known only few techniques for modification that may be generalized. Such techniques for modification capable of generalization includes amino acid substitution at one or two sites in VH to improve stability of a single chain antibody (scFv) as reported by Worn et. al. (Non-patent reference 2); amino acid substitution at one or plural sites in VH to improve an expression level of Fv fragments and to inhibit aggregation reaction as reported by Knappik et al. (Non-patent reference 3); amino acid substitution at one or two sites in VL to improve stability of a VL domain as reported by Steipe et al. (Non-patent reference 4); and amino acid substitution at position 34 (according to Kabat numbering) in VL to improve stability and an expression level of scFv as reported by Hugo et al. (Non-patent reference 5). However, the objective antibodies subject to modification by all these reports are mouse antibodies.
For the use as an antibody medicine, a mouse antibody is recognized as a foreign substance and excluded due to its high antigenicity when administered to human. Thus, it would be difficult to use a mouse antibody as a medicament for therapy of diseases. For dissolving this problem, a mouse monoclonal antibody may be converted to a chimeric antibody using a protein engineering technique. However, a mouse chimeric antibody still contains sequences derived from mice amounting to 30% or more and thus its repetitive or prolonged administration will lead to production of an antibody that inhibits the activity of the chimeric antibody administered to thereby not only extremely lower the efficacy of the medicament but to induce severe adverse side effects.
Therefore, main approach for solving these problems is to construct a humanized antibody wherein a complementarity determining regions (CDRs) from a mouse variable region are transplanted into a human variable region or a human antibody having sequences completely derived from human.
On the other hand, there is a report by Ewert et al. (Non-patent reference 6) in which amino acid substitution is done for a human antibody at one or plural sites in VH to allow for the improvement of stability and an expression level of scFv. However, the human antibody modified by Ewert et al. is of VH6 family and a frequency in use of the genes belonging to said family is as low as 1.4 to 2.4% (Non-patent reference 7), which renders their technique not being widely feasible one.
As described above, since a specific antibody targeting a disease-related antigen is extremely useful in the clinical field such as human diagnosis and therapy, establishment of the technique for preparing an antibody possessing all the features of high as specificity, low immunogenicity and high productivity has earnestly been desired.    Non-patent reference 1: Vriend et al., J. Comput. Aided Mol. Des., 7(4), p. 367-396 (1993)    Non-patent reference 2: Worn et al., Biochemistry, 37, p. 13120-13127 (1998)    Non-patent reference 3: Knappik et al., Protein Eng., 8(1), p. 81-89 (1995)    Non-patent reference 4: Steipe et al., J. Mol. Biol., 240, p. 188-192 (1994)    Non-patent reference 5: Hugo et al., Protein Eng., 16(5), p. 381-386 (2003)    Non-patent reference 6: Ewert et al., Biochemistry, 42, p. 1517-1528 (2003)    Non-patent reference 7: Brezinschek et al., J. Clin. Invest., 99(10), p. 2488-2501 (1997)